Human flavin-containing monooxygenase

ABSTRACT

A flavin-containing monooxygenase comprising the amino acid sequence of SEQ ID NO:8, a microorganism producing said flavin-containing monooxygenase, a flavin-containing monooxygenase gene containing a DNA sequence coding for the amino acid sequence of SEQ ID NO:8, a flavin-containing monooxygenase gene containing the DNA sequence of SEQ ID NO:7, a plasmid containing said flavin-containing monooxygenase gene, a microorganism containing said plasmid, and a method for producing the enzyme of the present invention wherein said microorganism is cultured to produce the enzyme.

This application is a Continuation in part of copending application Ser.No. 08/560,916 filed Oct. 20, 1995.

FIELD OF THE INVENTION

The present invention relates to a novel human flavin-containingmonooxygenase (FMO) gene.

DESCRIPTION OF THE PRIOR ART

Flavin-containing monooxygenases of rat, pig and rabbit are known asmicrosomal xenobiotic-metabolizing enzymes which oxidize variousxenobiotics including drugs, agricultural chemicals and environmentalpollutants. Some flavin-containing monooxygenases are characterized by aplurality of isozymes. Therefore, the precise characterization of andspecific function of each enzyme molecule are not understood well.Particularly with regard to human flavin-containing monooxygenases, only3 human flavin-containing monooxygenases have been elucidated by cDNAcloning (Dolphin et al., J. Biol. Chem., 266, 12379-12385, (1991),Dolphin et al., Biochem. J., 287, 261-267 (1992), Lomri et al. Proc.Natl. Acad. Sci. 89,1685-1689 (1992)). Therefore, many aspects of humanflavin-containing monooxygenases, such as population distribution andcharacterization of function to metabolize or to detoxify xenobiotics,remain unknown.

Under the circumstances, the present inventors found a novel humanflavin-containing monooxygenase and produced in a large quantity afunctional human flavin-containing monooxygenase by a DNA amplificationtechnique using particular oligonucleotides as primers.

SUMMARY OF THE INVENTION

Thus, the present invention provides nucleotide sequences encoding ahuman flavin-containing monooxygenases, wherein the nucleotide sequencesare RNA and DNA sequences, such as a flavin-containing monooxygenasegene containing a DNA sequence coding for the amino acid sequence of SEQID NO:7, a flavin-containing monooxygenase gene containing the DNAsequence of SEQ ID NO:7 (hereinafter, referred to as the gene of theinvention), a vector containing said flavin-containing monooxygenasegene (hereinafter, referred to as the vector of the invention), a hostcell containing said vector, and a method for producing the enzyme ofthe present invention, which comprises culturing said host cell andrecovering the enzyme produced thereby.

The host cell of the invention includes, but is not limited to,microorganisms such as yeast. The vector of the invention includes, butis not limited to, plasmid and phage vectors, such as a plasmid suitablefor expression in yeast. The present invention also provides nucleotidesequences that hybridize to the above-mentioned nucleotide sequences andthat encode an enzyme having the xenobiotic-oxidizing activity of ahuman flavin-containing monooxygenase. (By the term, "hybridization", itis intended to refer to conventional hybridization conditions andpreferably to stringent hybridization conditions.)

The present invention provides a novel human flavin-containingmonooxygenase, a host cell which produces said enzyme, a nucleotidesequence comprising said enzyme gene and nucleotide sequenceshybridizing therewith, and a vector containing said nucleotide sequence.The expressed enzyme of the present invention is useful in evaluatinghuman xenobiotic metabolism in vitro.

The present invention further provides an antibody, preferablymonoclonal, which specifically binds to an epitope of the enzyme of theinvention. The present invention further provides oligonucleotide probeswhich specifically bind the nucleotide sequence of the invention.

The present invention additionally provides a cell-free extract,preferably a microsomal fraction, prepared from a transformed host cellof the invention.

The present invention further provides a method for metabolizing asample compound comprising preparing a mixture of the sample compoundand the host cell or cell-free extract of the invention, incubating themixture and, optionally analyzing the products obtained thereby.

The above and other objects, features, and advantages of the presentinvention will be better understood from the following detaileddescriptions taken in conjunction with the accompanying drawing, whichis given by way of illustration only and is not limitative of thepresent invention.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 depicts a construction method of a yeast expression plasmid(pAFMO) for a human flavin-containing monooxygenase.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the present invention, a novel human flavin-containingmonooxygenase, antibodies thereto, a microorganism which produces saidenzyme, said enzyme gene, and a plasmid containing said gene areobtained. The expressed enzyme of the present invention is useful toevaluate xenobiotic metabolism.

The invention now will be described in detail hereinbelow.

The enzyme of the present invention is novel and differs from any of theaforementioned three known human flavin-containing monooxygenases. Theamino acid sequence of the instant enzyme or the DNA sequence coding forsaid polypeptide has about 50 to about 95% homology with the amino acidsequences or DNA sequences of the aforementioned known monooxygenases,respectively.

The gene of the present invention can be prepared by a conventionalgenetic engineering method, for example, cloning the subject gene from acDNA library. The cDNA library can be obtained by preparing the mRNAfraction of suitable cells, producing cDNA therefrom using reversetranscriptase and inserting said cDNA into a vector, preferably a phagevector or a plasmid vector. Alternatively, a commercially available cDNAlibrary derived from, for example, human liver can be employed. Thelibrary can be screened with either (i) a DNA fragment homologous to thegene or (ii) an antibody which binds to the instant enzyme. Furthermore,the gene can be prepared by amplifying and cloning the subject gene froma cDNA library by PCR using specific oligonucleotides as primers.

The DNA fragment for screening the library can be obtained by preparingdeduced oligonucleotides based on the amino acid sequence ofoligopeptides from the instant enzyme. Generally the deducedoligonucleotides comprise a family of possible sequences because of codedegeneracy, however, it is not impossible to find oligopeptide fragmentswhich would yield only one or two possible oligonucleotides which couldencode such amino acid fragments.

The resulting oligonucleotides then can be labelled by conventionalmethods and used to screen the cDNA library to obtain clones carryingnucleotide sequences which encode portions or all of the instant enzyme.

Alternatively, the cDNA library can be screened by an antibody whichbinds the instant enzyme. The antibody can be polyclonal or monoclonal,made by standard techniques known in the art. Thus, a suitable host,such as a rodent is immunized with the instant enzyme, or portionsthereof, using suitable diluents, routes and schedules as known in theart or which are optimized to obtain a suitable immune response thereto.At that point either serum, for polyclonal antisera, or splenocytes, formonoclonal antisera, are obtained.

Monoclonal antisera are developed using known techniques. The polyclonaland candidate monoclonal antibodies are screened for specificity, suchas in an ELISA using the instant enzyme as the solid-phase boundanalyte.

The relevant antibodies are used to screen the expression library. Theantibodies can be used as labelled reagents or as unlabelled reagents.If unlabeled, an additional step wherein a labelled ligand which bindsto the instant polyclonal or monoclonal antibody is used. Those reactionschemes will identify those clones which express protein which is boundby the instant antibodies specific for the instant enzyme.

Specific oligonucleotides which can be used as primers in a PCR includeDNA fragments as set forth in SEQ ID NO's:1-4. When said primers areused, about a 1.6 kb fragment corresponding to the protein coding regionof human flavin-containing monooxygenase gene, excluding 60 bp ofN-terminal sequences, can be amplified separately as two fragments ofabout 0.8 kb and about 1.0 kb. The resulting two fragments, of about 0.8kb and about 1.0 kb, and the fragment corresponding to the N-terminus ofabout 60 bp (linker) are ligated by a conventional genetic engineeringmethod, thereby resulting in the gene of the present invention.

The vector of the present invention usually is constructed by insertingthe gene of the present invention into an expression vector. Theexpression vector usually requires genetic information that enablesreplication in host microorganism cells, genetic information to enableindependent propagation, characteristics which ensure ready isolationand purification from the host microorganism cells and further adetectable marker. Such a plasmid can be constructed by a conventionalgenetic engineering method or is available commercially.

In a yeast expression system, for example, a yeast expression plasmidcan be prepared by inserting the gene of the present invention obtainedby cloning same into an expression vector containing a promoter andterminator which are functional in yeast.

As a functional promoter in yeast, examples include promoters of a yeastalcohol dehydrogenase gene (hereinafter, referred to as an ADHpromoter), of a glyceraldehyde-3-phosphate dehydrogenase gene(hereinafter, referred to as a GAPDH promoter) and of a phosphoglyceratekinase gene (hereinafter, referred to as a PGK promoter). The ADHpromoter can be prepared, for example, from the yeast expression vectorpAAH5 containing a yeast ADH1 promoter and terminator (available fromWashington Research Foundation, Ammerer et al. Meth. Enzymol., 101,192-201, U.S. Ser. No. 299,733 filed September 1991) by a conventionalgenetic engineering method. Said yeast expression plasmid containing theaforementioned promoter and terminator which are active in yeast and theinstant human flavin-containing monooxygenase gene can be constructedreadily by a conventional genetic engineering methods. For example, theplasmid can be constructed by inserting the instant humanflavin-containing monooxygenase gene into the HindIII sites of the yeastexpression vector pAAH5N containing the ADH promoter and ADH terminatordisclosed in JP-A No.21180/1990.

The resulting vector of the present invention is introduced into hostmicroorganism cells by a conventional method to produce a transformedhost cell. Then, said host cells are cultured to produce the enzyme ofthe present invention selectively and in large quantity.

For example, a plasmid of the present invention is introduced intoyeast, such as Saccharomyces cerevisiae, using known methods, such as analkali metal (LiCl) method. The yeast into which said plasmid isintroduced then is cultured by a conventional method to produce theenzyme of the present invention.

The recombinant vector and the polynucleotide of interest can be eitherRNA or DNA so long as the relevant polynucleotides can be used toidentify or to express the gene or enzyme of interest.

The expressed enzyme generally is localized in the microsomal membranewhere monooxygenase activity occurs. Therefore, the expressed enzymecan, for example, be used to investigate metabolism of a xenobiotic invitro, preferably in the form of intact yeast cells or cell-freeextracts. The xenobiotic can be a toxic substance, carcinogen ormutagen, or can be a compound which can be converted to a toxicsubstance, carcinogen or mutagen, for example, in vivo. Thus the instantenzyme can be used to test the xenobiotic or product thereof for anyadverse characteristics.

As the cell-free extract, for example, a microsomal fraction oftransformed cells can be used. Preparation of the cell-free extract orthe microsomal fraction may be performed according to a conventionalmethod described, for example, in DNA, 4(3): 203-210 (1985).

The yeast cells or the cell-free extract thus obtained may be used toanalyze a metabolic pathway of a sample compound by reacting the samplecompound with the yeast cells or the cell-free extract. The reaction canbe performed by adding the sample compound to a culture of the yeastcells or to a solution of cell-free extract, such as culture medium orbuffer containing the yeast cells or the cell-free extract, and byincubating the reaction mixture, for example, at a temperature of about10 to about 40° C., for about 0.1 to about 48 hours. The amount of theyeast cells or said cell-free extract and the amount of the samplecompound to be added to the reaction mixture may be varied according tovarious conditions such as the reaction temperature, reaction time andthe type of the sample compound. For example, the amount of the yeastcells or said cell-free extract is preferably between about 10⁷ andabout 10⁸ cells, or about 5 to about 200 μl of the microsomal fraction(per 1 ml of the solution), and the amount of the sample compound to beadded to the reaction mixture is preferably between about 0.01 and about1 μmole per 1 ml of the solution. The amounts optionally can beincreased or decreased as desired.

After completion of the reaction, analysis of the products andmetabolites in the reaction solution can be conducted according to aconventional analytical method, as described in Guideline ofInstrumental Analysis (New edition, first published 1985, KAGAKU-DOJINPublishing Company, edited by Jiro Shiokawa et al.) or SpectrometricIdentification of Organic Compounds (Fourth edition, third published1984, TOKYO KAGAKU DOJIN Co., Ltd., edited by R. M. Silver et al.). Asused herein, "metabolites" is meant to encompass any product resultingfrom an action of the instant enzyme.

Alternatively, the resulting products can be tested, for example, fortoxicity or transforming activity in standard assays and bioassays,using for example, cell lines and organisms as test systems for suchassessments. As used herein, "toxicity", is meant to encompass compoundswhich have a harmful effect on a cell, organism and the like. Thus, forexample, a carcinogen or a mutagen is toxic.

On the basis of the data obtained, it can be judged as to whether thesample compound either is detoxified or activated to a harmful compoundby the present enzyme.

The invention will be illustrated further with reference to thefollowing examples; however, the examples are not to be construed tolimit the scope of the invention.

EXAMPLE 1 Method for Obtaining the Gene of the Invention

Using the primers of SEQ ID NO's:1 to 4, about a 1.6 kb fragmentcorresponding to the protein coding region of human flavin-containingmonooxygenase without 60 bp of N-terminal sequence was amplifiedseparately as two fragments of about 0.8 kb and about 1.0 kb, using PCR.The amplified fragment of about 0.8 kb was cleaved with SacI andsubcloned into the SmaI-SacI site of the pUC A vector, which wasprepared by modifying the EcoRI site of pUC19 (TAKARA SHUZO) into aHindIII site, and by converting the cloning sites between the HindIIIsites into the following cloning sites:

    ______________________________________                                        HindIII XbaI ECGRI SpeI SphI PstI SalI BamHI SmaI KpnI SacI                   ______________________________________                                        HindII                                                                    

The obtained subclone was treated with SacI and ligated to the fragmentof about 1.0 kb which was pretreated with SacI, and then, the resultingplasmid was treated further with XbaI and XhoI into which the linkers ofSEQ ID NO's:5 and 6 were inserted (see FIG. 1). The gene correspondingto the protein coding region of human flavin-containing monooxygenasewas sequenced using a fluorescence DNA sequencer (model 373A, AppliedBiosystems) which is based on the dideoxy method. The result is shown inSEQ ID NO:7, accompanied by the deduced amino acid sequence (SEQ IDNO:8).

EXAMPLE 2 Construction of the Plasmid of the Invention

The gene corresponding to the protein coding region of humanflavin-containing monooxygenase was prepared by cleaving the obtainedplasmid with HindIII, and inserting same into pAAH5N, whereby the yeastexpression plasmid pAFMO was constructed, which allowed the gene of theinvention to be expressed in yeast (see FIG. 1).

EXAMPLE 3 Production of the Microorganism of the Invention

Saccharomyces cerevisiae AH22 was inoculated in 1 ml of YPD medium (1%(w/v) yeast extract, 2% (w/v) polypeptone, and 2% (w/v) glucose),cultivated at 30° C. for 18 hours, and then collected by centrifugation(10,000× g, 2 minutes, room temperature). The obtained cells weresuspended in 1 ml of 0.2 M LiCl solution, then centrifuged again, and tothe resulting pellet were added 20 μl of 1 M LiCl, 30 μl of 70% (w/v)polyethyleneglycol 4000 solution and 10 μl of the solution containingabout 1.0 μg of the plasmid of the present invention (pAFMO) constructedin Example 2. The resulting solution was mixed thoroughly, thenincubated at 30° C. for 1 hour, a further 140 μl of distilled water wasadded thereto and the mixture stirred. The solution was spread on the SDsynthetic plate (2.0% (w/v) glucose, 0.67% (w/v) yeast nitrogen basewithout amino acid, 20 g/ml histidine, 2.0% (w/v) agar), incubated at30° C. for 3 days, and the transformant containing the plasmid of thepresent invention (pAFMO) was obtained.

EXAMPLE 4 Preparation of the Microsomal Fraction of Yeast

The microorganism of the present invention (a transformed yeast producedin Example 3) was collected from 3.8 liters of liquid medium in whichthe microorganism was cultured up to a density of about 1.0×10⁸cells/ml. The yeast cells were suspended in 400 ml of buffer A (10 mMTris-HCl (pH 7.5), 2 M sorbitol, 0.1 mM DTT, 0.2 mM EDTA), and to theresulting solution was added 160 mg of Zymolyase 100T, and incubated at30° C. for 60 minutes. After suspending spheroplasts obtained bycentrifugation (5,000× g, 10 minutes, 4° C.) in 100 ml of buffer A, theresulting solution was subjected to centrifugation (5,000× g, 10minutes, 4° C.). After washing the spheroplasts by subjecting thesolution to centrifugation again under the same conditions, thespheroplasts were suspended in 200 ml of the buffer (10 mM Tris-HCl (pH7.5), 0.65 M sorbitol, 0.1 mM DTT), and disrupted by ultrasonication (50W, 5 minutes, 0° C.). The supernatant obtained by centrifugation (9,000×g, 20 minutes, 4° C.) was referred to as yeast S-9 Mix fractionhereinafter. The fraction was centrifuged further (125,000× g, 70minutes, 4° C.) to collect pellets, which then were suspended in 10 mlof 0.1 M phosphate buffer (pH 7.4) to obtain a microsomal fraction.

EXAMPLE 5 Assay of Thiourea S-oxygenase Activity in the MicrosomalFraction of Yeast Which Expresses the Enzyme of the Present Invention

The reaction was initiated by adding 200 μl of the microsomal fractionprepared in Example 4 and 25 μl of 120 mM thiourea to 2.5 ml of an assaysolution (0.1 M potassium phosphate buffer, pH 7.5, 0.2 mM NADPH, 160 μMthiocholine, 100 units catalase, 2 mM benzylimidazole, 0.4 mM EDTA)previously warmed at 37° C. Then, 400 μl of the mixture were added to 40μl of 3 M TCA every 3 minutes, the resulting mixture was left standingon ice, and then centrifuged to obtain 350 μl of a supernatant. To thesupernatant were added 1 ml of 1 M potassium phosphate buffer (pH 7.5),0.6 ml of water and 50 μl of 10 mM dithiobisnitrobenzoic acid forcoloration. The decrease of thiocholine was measured at an absorption of412 nm. Thiourea S-oxygenase activity was found in the yeast microsomalfraction expressing the present enzyme.

All references cited herein are incorporated by reference in entirety.

It will be evident that various changes and modifications can be madewithout deviating from the spirit and scope of the instant invention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 8                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #          31      GGAG CCCACCTGCT T                                          - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #          31      GCTG GGAGCTCATC A                                          - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #          31      TTTG GAACCTTCCT C                                          - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #          31      TCTG GGTATTGTCA G                                          - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 69 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - CTAGAATGGG GAAGAAAGTG GCCATCATTG GAGCTGGTGT GAGTGGCTTG GC - #CTCCATCA         60                                                                          #         69                                                                  - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 69 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - TTACCCCTTC TTTCACCGGT AGTAACCTCG ACCACACTCA CCGAACCGGA GG - #TAGTCCTC         60                                                                          #         69                                                                  - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1599 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1596                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - ATG GGG AAG AAA GTG GCC ATC ATT GGA GCT GG - #T GTG AGT GGC TTG GCC           48                                                                          Met Gly Lys Lys Val Ala Ile Ile Gly Ala Gl - #y Val Ser Gly Leu Ala           #                 15                                                          - TCC ATC AGG AGC TGT CTC GAG GAA GGA CTG GA - #G CCC ACC TGC TTT GAG           96                                                                          Ser Ile Arg Ser Cys Leu Glu Glu Gly Leu Gl - #u Pro Thr Cys Phe Glu           #             30                                                              - AAG AGC AAT GAC ATT GGG GGC CTG TGG AAA TT - #T TCA GAC CAT GCA GAG          144                                                                          Lys Ser Asn Asp Ile Gly Gly Leu Trp Lys Ph - #e Ser Asp His Ala Glu           #         45                                                                  - GAG GGC AGG GCT AGC ATT TAC AAA TCA GTC TT - #T TCC AAC TCT TCC AAA          192                                                                          Glu Gly Arg Ala Ser Ile Tyr Lys Ser Val Ph - #e Ser Asn Ser Ser Lys           #     60                                                                      - GAG ATG ATG TGT TTC CCA GAC TTC CCA TTT CC - #C GAT GAC TTC CCC AAC          240                                                                          Glu Met Met Cys Phe Pro Asp Phe Pro Phe Pr - #o Asp Asp Phe Pro Asn           # 80                                                                          - TTT ATG CAC AAC AGC AAG ATC CAG GAA TAT AT - #C ATT GCA TTT GCC AAA          288                                                                          Phe Met His Asn Ser Lys Ile Gln Glu Tyr Il - #e Ile Ala Phe Ala Lys           #                 95                                                          - GAA AAG AAC CTC CTG AAG TAC ATA CAA TTT AA - #G ACA TTT GTA TCC AGT          336                                                                          Glu Lys Asn Leu Leu Lys Tyr Ile Gln Phe Ly - #s Thr Phe Val Ser Ser           #           110                                                               - GTA AAT AAA CAT CCT GAT TTT GCA ACT ACT GG - #C CAG TGG GAT GTT ACC          384                                                                          Val Asn Lys His Pro Asp Phe Ala Thr Thr Gl - #y Gln Trp Asp Val Thr           #       125                                                                   - ACT GAA AGG GAT GGT AAA AAA GAA TCG GCT GT - #C TTT GAT GCT GTA ATG          432                                                                          Thr Glu Arg Asp Gly Lys Lys Glu Ser Ala Va - #l Phe Asp Ala Val Met           #   140                                                                       - GTT TGT TCT GGA CAT CAT GTG TAT CCC AAC CT - #A CCA AAA GAG TCC TTT          480                                                                          Val Cys Ser Gly His His Val Tyr Pro Asn Le - #u Pro Lys Glu Ser Phe           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - CCA GGA CTA AAC CAC TTT AAA GGC AAA TGC TT - #C CAC AGC AGG GAC TAT          528                                                                          Pro Gly Leu Asn His Phe Lys Gly Lys Cys Ph - #e His Ser Arg Asp Tyr           #               175                                                           - AAA GAA CCA GGT GTA TTC AAT GGA AAG CGT GT - #C CTG GTG GTT GGC CTG          576                                                                          Lys Glu Pro Gly Val Phe Asn Gly Lys Arg Va - #l Leu Val Val Gly Leu           #           190                                                               - GGG AAT TCG GGC TGT GAT ATT GCC ACA GAA CT - #C AGC CGC ACA GCA GAA          624                                                                          Gly Asn Ser Gly Cys Asp Ile Ala Thr Glu Le - #u Ser Arg Thr Ala Glu           #       205                                                                   - CAG GTC ATG ATC AGT TCC AGA AGT GGC TCC TG - #G GTG ATG AGC CGG GTC          672                                                                          Gln Val Met Ile Ser Ser Arg Ser Gly Ser Tr - #p Val Met Ser Arg Val           #   220                                                                       - TGG GAC AAT GGT TAT CCT TGG GAC ATG TTG CT - #C GTC ACT CGA TTT GGA          720                                                                          Trp Asp Asn Gly Tyr Pro Trp Asp Met Leu Le - #u Val Thr Arg Phe Gly           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - ACC TTC CTC AAG AAC AAT TTA CCG ACA GCC AT - #C TCT GAC TGG TTG TAC          768                                                                          Thr Phe Leu Lys Asn Asn Leu Pro Thr Ala Il - #e Ser Asp Trp Leu Tyr           #               255                                                           - GTG AAG CAG ATG AAT GCA AGA TTC AAG CAT GA - #A AAC TAT GGC TTG ATG          816                                                                          Val Lys Gln Met Asn Ala Arg Phe Lys His Gl - #u Asn Tyr Gly Leu Met           #           270                                                               - CCT TTA AAT GGA GTC CTG AGG AAA GAG CCT GT - #A TTT AAT GAT GAG CTC          864                                                                          Pro Leu Asn Gly Val Leu Arg Lys Glu Pro Va - #l Phe Asn Asp Glu Leu           #       285                                                                   - CCA GCA AGC ATT CTG TGT GGC ATT GTG TCC GT - #A AAG CCT AAC GTG AAG          912                                                                          Pro Ala Ser Ile Leu Cys Gly Ile Val Ser Va - #l Lys Pro Asn Val Lys           #   300                                                                       - GAA TTC ACA GAG ACC TCG GCC ATT TTT GAG GA - #T GGG ACC ATA TTT GAG          960                                                                          Glu Phe Thr Glu Thr Ser Ala Ile Phe Glu As - #p Gly Thr Ile Phe Glu           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - GGC ATT GAC TGT GTA ATC TTT GCA ACA GGG TA - #T AGT TTT GCC TAC CCC         1008                                                                          Gly Ile Asp Cys Val Ile Phe Ala Thr Gly Ty - #r Ser Phe Ala Tyr Pro           #               335                                                           - TTC CTT GAT GAG TCT ATC ATC AAA AGC AGA AA - #C AAT GAG ATC ATT TTA         1056                                                                          Phe Leu Asp Glu Ser Ile Ile Lys Ser Arg As - #n Asn Glu Ile Ile Leu           #           350                                                               - TTT AAA GGA GTA TTT CCT CCT CTA CTT GAG AA - #G TCA ACC ATA GCA GTG         1104                                                                          Phe Lys Gly Val Phe Pro Pro Leu Leu Glu Ly - #s Ser Thr Ile Ala Val           #       365                                                                   - ATT GGC TTT GTC CAG TCC CTT GGG GCT GCC AT - #T CCC ACA GTT GAC CTC         1152                                                                          Ile Gly Phe Val Gln Ser Leu Gly Ala Ala Il - #e Pro Thr Val Asp Leu           #   380                                                                       - CAG TCC CGC TGG GCA GCA CAA GTA ATA AAG GG - #A ACT TGT ACT TTG CCT         1200                                                                          Gln Ser Arg Trp Ala Ala Gln Val Ile Lys Gl - #y Thr Cys Thr Leu Pro           385                 3 - #90                 3 - #95                 4 -       #00                                                                           - TCT ATG GAA GAC ATG ATG AAT GAT ATT AAT GA - #G AAA ATG GAG AAA AAG         1248                                                                          Ser Met Glu Asp Met Met Asn Asp Ile Asn Gl - #u Lys Met Glu Lys Lys           #               415                                                           - CGC AAA TGG TTT GGC AAA AGC GAG ACC ATA CA - #G ACA GAT TAC ATT GTT         1296                                                                          Arg Lys Trp Phe Gly Lys Ser Glu Thr Ile Gl - #n Thr Asp Tyr Ile Val           #           430                                                               - TAT ATG GAT GAA CTC TCC TCC TTC ATT GGG GC - #A AAG CCC AAC ATC CCA         1344                                                                          Tyr Met Asp Glu Leu Ser Ser Phe Ile Gly Al - #a Lys Pro Asn Ile Pro           #       445                                                                   - TGG CTG TTT CTC ACA GAT CCC AAA TTG GCC AT - #G GAA GTT TAT TTT GGC         1392                                                                          Trp Leu Phe Leu Thr Asp Pro Lys Leu Ala Me - #t Glu Val Tyr Phe Gly           #   460                                                                       - CCT TGT AGT CCC TAC CAG TTT AGG CTG GTG GG - #C CCA GGG CAG TGG CCA         1440                                                                          Pro Cys Ser Pro Tyr Gln Phe Arg Leu Val Gl - #y Pro Gly Gln Trp Pro           465                 4 - #70                 4 - #75                 4 -       #80                                                                           - GGA GCC AGA AAT GCC ATA CTG ACC CAG TGG GA - #C CGG TCG TTG AAA CCC         1488                                                                          Gly Ala Arg Asn Ala Ile Leu Thr Gln Trp As - #p Arg Ser Leu Lys Pro           #               495                                                           - ATG CAG ACA CGA GTG GTC GGG AGA CTT CAG AA - #G CCT TGC TTC TTT TTC         1536                                                                          Met Gln Thr Arg Val Val Gly Arg Leu Gln Ly - #s Pro Cys Phe Phe Phe           #           510                                                               - CAT TGG CTG AAG CTC TTT GCA ATT CCT ATT CT - #G TTA ATC GCT GTT TTC         1584                                                                          His Trp Leu Lys Leu Phe Ala Ile Pro Ile Le - #u Leu Ile Ala Val Phe           #       525                                                                   #  1599            AA                                                         Leu Val Leu Thr                                                                   530                                                                       - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 532 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Met Gly Lys Lys Val Ala Ile Ile Gly Ala Gl - #y Val Ser Gly Leu Ala         #                 15                                                          - Ser Ile Arg Ser Cys Leu Glu Glu Gly Leu Gl - #u Pro Thr Cys Phe Glu         #             30                                                              - Lys Ser Asn Asp Ile Gly Gly Leu Trp Lys Ph - #e Ser Asp His Ala Glu         #         45                                                                  - Glu Gly Arg Ala Ser Ile Tyr Lys Ser Val Ph - #e Ser Asn Ser Ser Lys         #     60                                                                      - Glu Met Met Cys Phe Pro Asp Phe Pro Phe Pr - #o Asp Asp Phe Pro Asn         # 80                                                                          - Phe Met His Asn Ser Lys Ile Gln Glu Tyr Il - #e Ile Ala Phe Ala Lys         #                 95                                                          - Glu Lys Asn Leu Leu Lys Tyr Ile Gln Phe Ly - #s Thr Phe Val Ser Ser         #           110                                                               - Val Asn Lys His Pro Asp Phe Ala Thr Thr Gl - #y Gln Trp Asp Val Thr         #       125                                                                   - Thr Glu Arg Asp Gly Lys Lys Glu Ser Ala Va - #l Phe Asp Ala Val Met         #   140                                                                       - Val Cys Ser Gly His His Val Tyr Pro Asn Le - #u Pro Lys Glu Ser Phe         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Pro Gly Leu Asn His Phe Lys Gly Lys Cys Ph - #e His Ser Arg Asp Tyr         #               175                                                           - Lys Glu Pro Gly Val Phe Asn Gly Lys Arg Va - #l Leu Val Val Gly Leu         #           190                                                               - Gly Asn Ser Gly Cys Asp Ile Ala Thr Glu Le - #u Ser Arg Thr Ala Glu         #       205                                                                   - Gln Val Met Ile Ser Ser Arg Ser Gly Ser Tr - #p Val Met Ser Arg Val         #   220                                                                       - Trp Asp Asn Gly Tyr Pro Trp Asp Met Leu Le - #u Val Thr Arg Phe Gly         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Thr Phe Leu Lys Asn Asn Leu Pro Thr Ala Il - #e Ser Asp Trp Leu Tyr         #               255                                                           - Val Lys Gln Met Asn Ala Arg Phe Lys His Gl - #u Asn Tyr Gly Leu Met         #           270                                                               - Pro Leu Asn Gly Val Leu Arg Lys Glu Pro Va - #l Phe Asn Asp Glu Leu         #       285                                                                   - Pro Ala Ser Ile Leu Cys Gly Ile Val Ser Va - #l Lys Pro Asn Val Lys         #   300                                                                       - Glu Phe Thr Glu Thr Ser Ala Ile Phe Glu As - #p Gly Thr Ile Phe Glu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Gly Ile Asp Cys Val Ile Phe Ala Thr Gly Ty - #r Ser Phe Ala Tyr Pro         #               335                                                           - Phe Leu Asp Glu Ser Ile Ile Lys Ser Arg As - #n Asn Glu Ile Ile Leu         #           350                                                               - Phe Lys Gly Val Phe Pro Pro Leu Leu Glu Ly - #s Ser Thr Ile Ala Val         #       365                                                                   - Ile Gly Phe Val Gln Ser Leu Gly Ala Ala Il - #e Pro Thr Val Asp Leu         #   380                                                                       - Gln Ser Arg Trp Ala Ala Gln Val Ile Lys Gl - #y Thr Cys Thr Leu Pro         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ser Met Glu Asp Met Met Asn Asp Ile Asn Gl - #u Lys Met Glu Lys Lys         #               415                                                           - Arg Lys Trp Phe Gly Lys Ser Glu Thr Ile Gl - #n Thr Asp Tyr Ile Val         #           430                                                               - Tyr Met Asp Glu Leu Ser Ser Phe Ile Gly Al - #a Lys Pro Asn Ile Pro         #       445                                                                   - Trp Leu Phe Leu Thr Asp Pro Lys Leu Ala Me - #t Glu Val Tyr Phe Gly         #   460                                                                       - Pro Cys Ser Pro Tyr Gln Phe Arg Leu Val Gl - #y Pro Gly Gln Trp Pro         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Gly Ala Arg Asn Ala Ile Leu Thr Gln Trp As - #p Arg Ser Leu Lys Pro         #               495                                                           - Met Gln Thr Arg Val Val Gly Arg Leu Gln Ly - #s Pro Cys Phe Phe Phe         #           510                                                               - His Trp Leu Lys Leu Phe Ala Ile Pro Ile Le - #u Leu Ile Ala Val Phe         #       525                                                                   - Leu Val Leu Thr                                                                 530                                                                       __________________________________________________________________________

What we claim is:
 1. An isolated enzyme having xenobiotic oxidizingactivity of human flavin-containing monooxygenase and a polypeptidesequence of SEQ ID NO:8.